Rapid and visual detection of berberine hydrochloride based on a water‐soluble pyrene derivative

In this study, a rapid method for the detection of berberine hydrochloride (BRH) was developed based on a water‐soluble pyrenyl probe, 8‐hydroxypyrene‐1,3,6‐trisulfonic acid (HPTS). This method features low cost, good selectivity, high sensitivity and a fast response. The sensing mechanism of this probe is attributed to the formation of a complex between HPTS and BRH induced by electrostatic interaction and π–π stacking. To the best of our knowledge, this is the first fluorescent sensor for BRH based on organic materials that has low cost and a visual response. The detection limit of this method was as low as 1.24 μM and the linear response range is 2–50 μM. This method also allowed rapid detection of BRH real samples.     

A new sensitive, rapid fluorescence technique for the determination of proteins in gel electrophoresis and in solution

We describe the use of o-phthalaldehyde (OPT) in a simple, rapid, fluorimetric technique for the proteins in the concentration range of 0.1–50 μg/ml. Fluorescence excitation is near 338 nm and emission is measured near 446 nm.The technique is eminently suitable for simple rapid, high-resolution evaluation of protein/peptide separations in polyacrylamide gel electrophoresis. For this purpose the mixture to be fractionated is treated first with β-mercaptoethanol, followed by OPT, prior to electrophoresis. The separation is evaluated after electrophoresis by exciting the fluorescence of protein/peptide-bound OPT with a conventional (365 nm) “black light”. The technique provides exceptional resolution and far greater sensitivity than conventional detection techniques; its detection limits are ca. 10 ng protein. When used without β-mercaptoethanol, the technique could also allow definition of the distribution of SH-containing proteins/peptides.Quantitative evaluation of protein/peptide distribution is best achieved by photometric scanning of photographic negatives obtained under defined exposure and developing times.     

Multiplexed DNA detection using a gold nanorod‐based fluorescence resonance energy transfer technique

A fluorescence resonance energy transfer method for multiplex detection DNA based on gold nanorods had been successfully constructed. This method is simple, easy to operate, good selectivity, no requirement to label the probe molecule and can analyze simultaneously multiple targets of DNA in one sample. The limit of detection for the 18‐mer, 27‐mer and 30‐mer targets is 0.72, 1.0 and 0.43 nM at a signal‐to‐noise ratio of 3. The recoveries of three targets were 96.57–98.07%, 99.12–100.04% and 97.29–99.93%, respectively. The results show that the method can be used to analyze a clinical sample or a biological sample; it also can be used to develop new probes for rapid, sensitive and highly selective multiplex detection of analytes in real samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of the major constituents in fruit of Arctium lappa L. by matrix solid-phase dispersion extraction coupled with HPLC separation and fluorescence detection

The arctiin and arctigenin in the fruit of Arctium lappa L. were extracted by matrix solid-phase dispersion (MSPD) and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The experimental conditions for the MSPD were optimized. Silica gel was selected as dispersion adsorbent and methanol as elution solvent. The calibration curve showed good relationship (r > 0.9998) in the concentration range of 0.010–5.0 μg mL⁻¹ for arctiin and 0.025–7.5 μg mL⁻¹ for arctigenin. The recoveries were between 74.4% and 100%. The proposed method consumed less sample, time and solvent compared with conventional methods, including ultrasonic and Soxhlet extraction.     

Rapid and sensitive gas-chromatographic determination of caffeine in blood plasma,saliva, and xanthine beverages

A gas chromatographic procedure is reported for the determination of caffeine in plasma, saliva, and xanthine beverages. Using a 75 cm column packed with OV-17, nitrogen-sensitive detection, and 1 ml samples, a suitable limit of analysis (coefficient of variation (CV)=10.2%) of 50 ng/ml was obtained in plasma. Within-day CVs at caffeine concentrations of 0.1–0.5–2.0–7.5–15.0 g/ml in plasma were 7.7–5.6–4.8–3.8–3.4%, respectively. The limit of detection, defined as the injected quantity of caffeine giving rise to a signal to noise ratio of 2, is 40 pg, corresponding to a plasma concentration of 1 ng/ml.The procedure involves addition of the internal standard 7-pentyl theophylline and alkaline extraction of the sample with dichloromethane. The method described rivals any gaschromatographic assay published so far in rapidness and accuracy.Plasma and saliva caffeine concentrations were determined in a healthy male volunteer after swallowing 400 ml of coffee. The calculated pharmacokinetic parameters, assuming complete absorption of caffeine from the G.I. tract, agree well with previously published values.     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum

Current clinically assays, such as enzyme‐linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high‐throughput analysis. A novel assay based on magnetic beads and time‐resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti‐HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium‐labeled anti‐HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02–700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra‐ and interassay coefficients of variation were 4.7–8.7% and 3.8–7.5%, respectively. The performance of this assay was further assessed against a well‐established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X – 0.017, R = 0.989). In the current study, we demonstrated that this novel time‐resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high‐throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel fluorescent indicator-based fluorene derivative for rapid and visual trace water sensing

Fluorene nucleus derivatives show great potential for building outstanding fluorescence probes. In this paper, a novel fluorescent probe was developed by reacting with fluorene core with azacyclobutane, which exhibits typical solvation chromogenic effect in solvent. The fluorescence of the probe quenched in highly polar solvent. Based on this phenomenon, a novel fluorescence system for trace water was constructed. The response of this probe was fast (30 s) and sensitive for the detection of trace water in organic solvents, and the detection limit of water content in DMSO reached 0.13%. In addition, the probe can also be made as a test strip combined with homemade portable device and a smartphone for rapid detection of trace water. The luminescence mechanism of the probe is theoretically calculated based on time-contained density functional theory (TDDFT). To showcase its practicality, it has been applied for the detection of trace water in honey and alcohol by dipstick. This method provides a new idea for designing efficient fluorescent probes based on dipstick and mobile phone rapid detection.     

Rapid determination of hazardous compounds in food based on a competitive fluorescence microsphere immunoassay

Development of a microsphere-based competitive fluorescence immunoassay for the determination of hazardous low-molecular-weight compounds in food is described. In this method, antigens are covalently bound to carboxy-modified microspheres to compete monoclonal antibody with low-molecular-weight compounds in food samples; mouse IgG/fluorescein isothiocyanate conjugate is used as the fluorescent molecular probe. Thus, the hazardous low-molecular-weight compounds are quantified using a multiparameter flow cytometer. This method has been evaluated using clenbuterol as a model compound. It has a sensitivity of 0.01 ng/mL with dynamic range of 0.01-100 ng/mL, and the concentration of clenbuterol providing 50% inhibition (IC₅₀) is 1.1 ng/mL. The main advantages of this method are its high efficiency, biocompatibility, and selectivity, as well as ultralow trace sample consumption and low cost.     

A simple and sensitive fluorescence method for the determination of trace ozone in air using acridine red as a probe

The ozone in an air sample was trapped by H₃BO₃‐LK solution to produce iodine (I₂) that interacted with excess I^– to form I₃^–. In pH 4.0 acetate buffer solutions, the I₃^– reacted with acridine red to form acridine red–I₃ ion association particles that resulted in the fluorescence peak decreased at 553 nm. The decreased value ΔF_{553 nm} is linear to the O₃ concentration in the range 0.08–53.3 × 10^–6 mol/L, with a detection limit of 4 × 10^–8 mol/L. This fluorescence method was used to determine ozone in air samples, and the results were in agreement with that of indigo carmine spectrophotometry. Copyright © 2014 John Wiley & Sons, Ltd.     

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.     

Simple and rapid analysis of tocilizumab using HPLC‐fluorescence detection method

We developed a novel assay using high‐performance liquid chromatography (HPLC) with fluorescence detection for the determination of tocilizumab (TCZ), after it has undergone a facile and rapid pretreatment. TCZ belongs to the same subclass as IgG1 (Immunoglobulin G subclass 1), and we could separate TCZ from IgG1 without antigen–antibody reactions, with the novel detection method. The separation of these antibodies was achieved by pretreatment with an organic solvent containing a base, such as trimethylamine and triethylamine. The effect of these bases on the separation of TCZ is related to the hydrophobicity of the base rather than the electrostatic charge. The results indicated that the surface charge of antibodies changed because of the structural change, even though the difference in the amino acid sequences of the antibodies was very low. Our method is available for the separation of the antibody subclasses, and it would be useful to assay TCZ in blood.     

Chlorophyll fluorescence technique as a rapid diagnostic test of the effects of the photosynthetic inhibitor chlorotoluron on two winter wheat cultivars

A modified chlorophyll fluorescence technique was evaluated as a rapid diagnostic test of the susceptibility of wheat cultivars to chlorotoluron. Two winter wheat cultivars (Maris Huntsman and Mercia) exhibited differential response to the herbicide. All of the parameters of chlorophyll fluorescence examined were strongly influenced by herbicide concentration. Additionally, the procedure adopted here for the examination of winter wheat cultivar sensitivity to herbicide indicated that the area above the fluorescence induction curve and the ratio F_V/F_M are appropriate chlorophyll fluorescence parameters for detection of differential herbicide response between wheat cultivars. The potential use of this technique as an alternative to traditional methods of screening new winter wheat cultivars for their response to photosynthetic inhibitor herbicide is demonstrated here.     

A simple and sensitive label‐free fluorescence sensing of heparin based on Cdte quantum dots

A rapid, simple and sensitive label‐free fluorescence method was developed for the determination of trace amounts of an important drug, heparin. This new method was based on water‐soluble glutathione‐capped CdTe quantum dots (CdTe QDs) as the luminescent probe. CdTe QDs were prepared according to the published protocol and the sizes of these nanoparticles were verified through transmission electron microscopy (TEM), X‐ray diffraction (XRD) and dynamic light scattering (DLS) with an average particle size of about 7 nm. The fluorescence intensity of glutathione‐capped CdTe QDs increased with increasing heparin concentration. These changes were followed as the analytical signal. Effective variables such as pH, QD concentration and incubation time were optimized. At the optimum conditions, with this optical method, heparin could be measured within the range 10.0–200.0 ng mL^?1 with a low limit of detection, 2.0 ng mL^?1. The constructed fluorescence sensor was also applied successfully for the determination of heparin in human serum. Copyright © 2015 John Wiley & Sons, Ltd.     

基于叶绿素荧光研究球形棕囊藻在富磷条件下的生长特性

棕囊藻属于定鞭藻纲、定鞭藻目,广泛分布在不同海洋生态环境中。有报道指出,棕囊藻时常在北太平洋温带港湾、挪威海、北海、英吉利海峡及南极海域等地方引发的大规模有害赤潮。近年来,我国东海海域和南海粤东海域均发生较大面积的棕囊藻赤潮,给当地的水产养殖业带来了严重的影响。除此之外,棕囊藻具有特殊的生理机制,可以产生二甲基硫化物(DMS),对整个海域的气候状况,酸雨酸雾的形成以及全球硫循环都有重要的意义。本文以球形棕囊藻为研究材料,设置3组较高的磷浓度处理(5mg·L^-1、10mg·L^-1和20mg·L^-1),利用细胞记数和叶绿素荧光测定等方法研究了该藻在不同富磷浓度下的生长情况。结果显示,不同磷浓度下的藻体荧光值变化均呈现"S"型曲线,表明藻细胞的生长经历缓慢期,快速期和平缓期3个阶段;同时,试验所设置的磷浓度对球形棕囊藻的叶绿素荧光值有一定的影响,其中在5mg·L^-1磷浓度下的藻体荧光值最低,在第7天只有802μg·L^-1,而在10mg·L^-1和20mg·L^-1磷浓度下的藻体荧光值较高,在第7天分别达到836μg·L^-1和850μg·L^-1,表明磷营养可以促进藻细胞的生长增殖,但在较高磷浓度下,这种促进作用不明显。结果还显示,较低浓度的磷(5mg·L^-1)减缓与限制了藻细胞的生长,在5mg·L^-1磷浓度下的藻最大特定比生长速率和细胞密度分别只有0.704d^-1和190cells·mL^-1。相对而言,20mg·L^-1磷浓度下的藻最大特定比生长速率和细胞密度最高,分别达到了0.771d^-1和250cells·mL^-1。研究结果揭示,水体中的磷营养浓度的变化是导致藻细胞大量增殖的一个主要的外在因素,而利用叶绿素荧光来测定藻细胞生长是一种快速、简便和可靠的方法,在今后有害水华监测过程中应该多加利用,以更及时、准确地预测预报有害水华的发生,降低其对经济、环境和社会造成的潜在危害。     

Application of 2-halopyridinium salts as ultraviolet derivatization reagents and solid-phase extraction for determination of captopril in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of captopril in human plasma. 1-Benzyl-2-chloropyridinium bromide (BCPB) was used as a precolumn derivatizing reagent. The mercapto group of captopril was arylated by the reagent to generate a stable UV-sensitive product. The derivative was solid-phase extracted (SPE), separated on a C₁₈ column using reversed-phase ion-paring chromatography and monitored by a spectrophotometric detector at 314 nm. The method enabled sensitive determination of captopril and its disulphides in human plasma in patients after oral administration. Disulphides of captopril with captopril itself and with endogenous thiol compounds are reduced with triphenylphosphine to form captopril, followed by derivatization with the same reagent. The quantification limit was 10 ng/ml. Calibration curves were prepared for human plasma samples spiked with captopril and captopril disulphide. The calibration curves were linear in the range of 10 to 500 ng/ml for captopril and 10 to 1000 ng/ml for captopril disulphide.     

Highly sensitive fluorescence detection of glycoprotein based on energy transfer between CuInS2 QDs and rhodamine B

A highly sensitive fluorescence method for glycoprotein detection has been established based on fluorescence resonance energy transfer (FRET) between CuInS₂ quantum dots (QDs) and rhodamine B (RB). Lectins comprise a group of proteins with unique affinities toward carbohydrate structures, so the process of FRET can occur between lectin‐coated QDs (CuInS₂ QDs–Con A conjugates, acceptors) and carbohydrate‐coated RB (RB–NH₂‐glu conjugates, donors). The fluorescence of lectin‐coated QDs was recovered in the presence of a glycoprotein such as glucose oxidase (GOx) and transferrin (TRF), which significantly reduced the FRET efficiency between the donor and the acceptor. Under optimal conditions, a linear correlation was established between the fluorescence intensity ratio I₆₅₄/I₅₇₇ and the TRF concentration over the range of 6.90 × 10^‐10 to 3.45 × 10^‐8 mol/L, with a detection limit of 2.5 × 10^‐10 mol/L. The linear range for GOx is 3.35 × 10^‐10 to 6.70 × 10^‐8 mol/L, with a detection limit of 1.5 × 10^‐10 mol/L. The proposed method was applied to the determination of glycoprotein in human serum and cell‐extract samples with satisfactory results. Furthermore, CuInS₂ QDs–Con A conjugates are used as safe and efficient optical nanoprobes in HepG2 cell imaging. Copyright © 2015 John Wiley & Sons, Ltd.     

Synchronous fluorescence determination of phytic acid in FOODSTUFFS and urine based on replacement reaction

Introduction – Phytic acid is a ubiquitous and abundant natural component in many plant seeds, fruits and vegetables. Its biological and pharmaceutical functions are still controversial. The examination on the level of phytic acid in foodstuffs and urine can provide valuable information for its dietary intake and metabolism. Objective – To develop a sensitive and reliable synchronous fluorescence protocol for determination of phytic acid in selected foodstuffs and human urine. Methodology – Phytic acid efficiently catches Cu²⁺ ion in previously prepared Cu^II‐2,2′‐bipyridine complex in aqueous solution, releasing the fluorescent 2,2′‐bipyridine molecule and recovering synchronous fluorescence. The recovered fluorescence is proportional to the added phytic acid, by which the levels of phytic acid in the selected foodstuffs and human urine are quantified. Results – A calibration curve with a regression equation of I_f = 37.745 + 39.245c (R² > 0.9988) showed good linearity over the range 0.18–17.50 mg/L phytic acid. The relative standard deviation at 95% confidence degree was less than 2.04% (n = 5), indicating that the procedures are reproducible. The detection and quantification limit of phytic acid were estimated to be 0.12 and 0.18 mg/L, respectively. By the proposed method, phytic acid in the selected foodstuffs and urine was determined to be 3.25–16.76 and 0.43–1.21 mg/L with recoveries of 96.8%–105.6% and 95.1%–104.2%, respectively. The results are in good agreement with those obtained by the reported HPLC technique. Conclusion – The developed method is sensitive, reliable and economical, which permits its practical application in quantitative analyses of trace phytic acid in foodstuffs and urine. Copyright © 2010 John Wiley & Sons, Ltd.